The present invention relates generally to electroporation systems and more particularly to systems and methods for stabilizing resistance measurements and for controlling the duration of applied electrical pulses in electroporation systems.
It is known that exposure of cells to intense electric fields for brief periods of time temporarily destabilized membranes. This effect has been described as a dielectric breakdown due to an induced transmembrane potential, and has been termed xe2x80x9celectroporationxe2x80x9d. A variety of procedures use electroporation treatments including production of monoclonal antibodies, cell-cell fusion, cell-tissue fusion, insertion of membrane proteins, and genetic transformation. Protocols for the use of electroporation to load cells in vitro typically use a suspension of single cells or cells that are attached in a planar manner to a growth surface. In vivo electroporation is more complex because tissues are involved. Tissues are composed of individual cells that collectively make up a three-dimensional structure. In either case, the effects on the cell are generally the same.
The cells or tissue are exposed to electric fields by administering one or more direct current pulses. Electrical treatment is conducted in a manner that results in a temporary membrane destabilization with minimal cytotoxicity. The intensity of electrical treatment is typically described by the magnitude of the applied electric field. This field is defined as the voltage applied to the electrodes divided by the distance between the electrodes. Electric field strengths ranging from 1000 to 5000 V/cm are typically used for delivering molecules in vivo and are also specific to the cells or tissue under investigation.
U.S. Pat. Nos. 6,258,592, 5,729,426, 5,656,926, 5,642,035, and 4,750,100, each of which are hereby incorporated by reference in its entirety, disclose examples of electroporation systems and various subsystems such as voltage control subsystems. In a typical electroporation system, an internal charge reservoir (e.g., a capacitor) stores electric energy (charge). A high-voltage switch couples the charge reservoir to a sample cuvette containing cells to be electroporated, and the stored charge discharges into the sample, typically as one or more pulses. Pulses are usually exponentially decaying in shape; however, square waves have also been used. The duration of each pulse is called pulse width. Molecule loading has been performed with pulse widths ranging from microseconds (xcexcs) to milliseconds (ms). Typically, one or multiple pulses are utilized during electrical treatment.
One problem is that the sample can have a wide range of resistance values, however, the electroporation effect may have a narrow range of parameters, e.g., pulse voltage and pulse duration, for which efficiency is highest. There is a narrow gap between electroporation and electrocution; if a pulse has too long a duration or too high a field strength, the cells may be lysed (destroyed). Minimal conditions tend to reduce the efficiency of electroporation. It is, therefore, desirable to provide systems and methods for controlling the pulse duration, or time constant, of electroporation pulses.
It is also generally desirable to apply an appropriate voltage to the sample so as to not damage the sample. Thus, it is important to accurately determine the resistance of the sample. Methods and apparatus for measuring resistance in electroporation systems and correcting for the series protection resistor are disclosed in U.S. Pat. Nos. 5,729,426, 5,642,035, and 5,656,926, each previously incorporated by reference in its entirety for all purposes. However, the resistance-measuring circuits as disclosed therein require sufficient stability of a coupling capacitor to achieve high-accuracy measurements.
Accordingly, it is desirable to provide systems, methods and apparatus for stabilizing resistance measurements in electroporation systems to achieve high-accuracy measurements and to provide systems and methods for controlling the pulse duration, or time constant, of electroporation pulses.
The present invention provides systems, methods and apparatus for stabilizing resistance measurements in electroporation systems so as to achieve high-accuracy measurements. The present invention also provides systems, methods and apparatus for controlling the pulse duration, or time constant, of electroporation pulses.
According to the present invention, circuitry is provided to accurately determine the resistance of a sample in the electroporation system, for example a sample provided in an electroporation cuvette. Additionally, a resistance control system is provided to automatically measure the resistance of a sample, determine a capacitance and determine a parallel add-on resistance that substantially provides a desired pulse duration (time constant) for an electroporation pulse.
According to an aspect of the invention, a method is provided for applying an electrical pulse to a sample for a desired duration. The method typically includes identifying a desired pulse duration, automatically measuring the resistance Rs of the sample, automatically selecting a capacitance C to be placed in series with the sample, and automatically determining a resistance R2 to be placed in parallel with the sample such that a time constant T=Cxc3x97R is substantially equal to the desired pulse duration, wherein R=(Rsxc3x97R2)/(Rs+R2). In certain aspects, R2 may be implemented using a single resistor or a multiple resistor arrangement, however, R2 is generally a composite resistance that includes any resistance continuously within the circuit arrangement and any selected component or components. Similarly, C may be a composite capacitance including any capacitance continuously within the circuit arrangement and any selected component or components.
According to another aspect of the present invention, a method is provided for automatically determining the resistance of a sample in an electroporation system. The method typically includes applying a first input signal to the sample across a coupling capacitor and a drive resistor circuit, the first input signal having an input voltage Ein at a first frequency f1, the drive resistor circuit having a resistance R1, and measuring the voltage E1 across the sample at said first frequency. The method also typically includes applying a second input signal to the sample across the drive resistor circuit, the second input signal having the input voltage Ein at a second frequency f2, and measuring the voltage E2 across the sample at said second frequency. The method further typically includes determining the sample resistance Rs using Ein and the measured values E1 and E2 without using the capacitance of the coupling capacitor.
According to yet another aspect of the invention, a circuit arrangement is provided in an electroporation system configured to determine the resistance of a sample. The arrangement typically includes a signal source for providing a sinusoidal voltage signal to a sample across a coupling capacitor and drive resistor, a frequency switch coupled to the signal source, the switch configured to adjust the frequency of the voltage signal applied by the signal source, and a microprocessor coupled to the frequency switch, an output of the signal source and the sample. The microprocessor typically controls the signal source, via the frequency switch, to apply a first voltage signal to the sample at a first frequency and a second voltage signal to the sample at a second frequency, the first and second voltage signals having substantially the same voltage amplitude. The microprocessor is typically configured to determine the sample resistance Rs based on the applied voltage amplitude of the first and second voltage signals and on the detected voltage levels across the sample at the first and second frequencies without using the capacitance of the coupling capacitor.
Reference to the remaining portions of the specification, including the drawings claims and Appendices, will realize other features and advantages of the present invention. Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with respect to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements.